Assays and methods for the diagnosis of post-streptococcal disorders

ABSTRACT

Provided are methods for diagnosing a disease in a subject with a previous streptococcal infection by determining the presence or absence of one or more autoantibodies in a biological sample from the subject, wherein the one or more autoantibodies recognize an antigen from a protein selected from the group consisting of ELAVL2, ELAVL3, ELAVL4, Nova-1, Nova-2, Cdr1, Cdr2; and Cdr3. The presence of such autoantibodies is indicative of a positive diagnosis for a post-streptococcal disease such as PANDAS, post-GABHS glomerulonephritis, rheumatic fever, autism and Syndenham&#39;s chorea.

FIELD OF THE INVENTION

The invention relates to medically useful assays and methods for thediagnosis of post-streptococcal infection complications includingPANDAS, post-GABHS glomerulonephritis, rheumatic fever, and Syndenham'schorea

BACKGROUND OF THE INVENTION

Pediatric Autoimmune Neuropsychiatric Disorder Associated with group AStreptococcal infection (PANDAS) is characterized by pediatric onset;neuropsychiatric disorders (obsessive compulsive disorder [OCD]) and/ortic disorder; abrupt onset and/or episodic course of symptoms;association with group A hemolytic streptococcal (GABHS) infection; andassociation with neurological abnormalities (motor hyperactivity oradventitious movements, including choreiform movements or tics). PANDASis one of a spectrum of post-streptococcal infection complications thatinclude post-GABHS glomerulonephritis, rheumatic fever, and Syndenham'schorea.

According to the National Institute of Mental Health (NIMH), childrenwith PANDAS are clinically identified by certain criteria. Thesecriteria include: (1) presence of obsessive-compulsive disorder and/or atic disorder; (2) pediatric onset of symptoms (age 3 years to puberty);(3) episodic course of symptom severity; (4) association with group Astreptococcal infection; and (5) association with neurologicalabnormalities (motoric hyperactivity, or adventitious movements, such aschoreiform movements).

The children usually have dramatic, “overnight” onset of symptoms,including motor or vocal tics, obsessions, and/or compulsions. Somestudies have shown no acute exacerbations associated with streptococcalinfections among clinically defined PANDAS subjects while others haveshown a profound one.

In addition to an OCD or tic disorder diagnosis, children may have othersymptoms associated with exacerbations such as emotional lability,enuresis, anxiety, and deterioration in handwriting. Because themolecular mechanisms underlying the disease are largely unknown, thereis no clear diagnostic test for these diseases. Thus, there is a need inthe art for methods and assays capable of diagnosing PANDAS and otherpost-streptococcal infection complications.

SUMMARY OF THE INVENTION

The instant invention is based on the discovery that an immune reactionto a streptococcal infection can result in the production ofautoantibodies capable of reacting with neuronal cells and inducing apathological condition.

In a first aspect, the invention provides a method for diagnosing adisease in a subject, said method comprising,

(a) determining the presence or absence of one or more autoantibodies ina biological sample from the subject with a previous Streptococcalinfection, wherein the one or more autoantibodies recognize an antigenfrom a protein selected from the group consisting of ELAVL2, ELAVL3,ELAVL4, Nova-1, Nova-2, Cdr1, Cdr2 and Cdr3.

(b) identifying the subject as having a disease selected from the groupconsisting of PANDAS, post-GABHS glomerulonephritis, rheumatic fever,autism spectrum disorders and Syndenham's chorea, when the presence ofone or more autoantibodies is detected, and identifying the subject asnot having one of said diseases caused by the Streptococcal infectionwhen said autoantibodies are absent.

In another aspect, the invention provides for a method comprising:

(a) testing a biological sample from a subject for the presence of amarker of a streptococcal infection; and

(b) testing the biological sample for the presence or absence of one ormore autoantibodies that recognize an antigen from a protein selectedfrom the group consisting of ELAVL2, ELAVL3, ELAVL4, Nova-1, Nova-2,Cdr1, Cdr2 and Cdr3.

In yet another aspect, there is provided a method comprising:

(a) testing a biological sample from a subject for the presence of amarker of a streptococcal infection; and

(b) testing the reactivity of one or more autoantibodies in the sampleto neuronal cells wherein the one or more autoantibodies recognize anantigen from a protein selected from the group consisting of ELAVL2,ELAVL3, ELAVL4, Nova-1, Nova-2, Cdr1, Cdr2 and Cdr3.

In one embodiment of the above aspects, testing a biological sample froma subject for the presence of a marker of a streptococcal infectioncomprises determining the presence or absence of one or moreanti-streptococcal antibodies. As used herein, the terms “antibody,”“antibodies,” “autoantibody,” and “autoantibodies” includes wholeantibodies and any antigen binding fragment or a single chain thereof.Thus the term “antibody” includes any protein or peptide containingmolecule that comprises at least a portion of an immunoglobulinmolecule. The terms “antibody,” “antibodies,” “autoantibody,” and“autoantibodies” also include immunoglobulins of any isotype, fragmentsof antibodies which retain specific binding to antigen, including, butnot limited to, Fab, Fab', F(ab)₂, Fv, scFv, dsFv, Fd fragments, dAb,VH, VL, VhH, and V-NAR domains; minibodies, diabodies, triabodies,tetrabodies and kappa bodies; multispecific antibody fragments formedfrom antibody fragments and one or more isolated. Examples of suchinclude, but are not limited to a complementarity determining region(CDR) of a heavy or light chain or a ligand binding portion thereof, aheavy chain or light chain variable region, a heavy chain or light chainconstant region, a framework (FR) region, or any portion thereof. Thevariable regions of the heavy and light chains of the immunoglobulinmolecule contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies (Abs) may mediate the binding of theimmunoglobulin to host tissues. The term “anti-” when used before aprotein name, anti-Ri, anti-Yo, and anti-Hu, for example, refers to amonoclonal or polyclonal antibody that binds and/or has an affinity to aparticular antigen. For example, “anti-Hu” refers to an antibody thatbinds to the Hu antigen. The antibodies can be polyclonal, monoclonal,multispecific (e.g., bispecific antibodies), and antibody fragments, solong as they exhibit the desired biological activity.

The terms “antigen” and “antigenic” refer to molecules with the capacityto be recognized by an antibody or otherwise act as a member of anantibody-ligand pair. “Specific binding” refers to the interaction of anantigen with the variable regions of immunoglobulin heavy and lightchains. Antibody-antigen binding may occur in vivo or in vitro. Theskilled artisan will understand that macromolecules, including proteins,nucleic acids, fatty acids, lipids, lipopolysaccharides andpolysaccharides have the potential to act as an antigen. The skilledartisan will further understand that nucleic acids encoding a proteinwith the potential to act as an antibody ligand necessarily encode anantigen. The artisan will further understand that antigens are notlimited to full-length molecules, but can also include partialmolecules. The term “antigenic” is an adjectival reference to moleculeshaving the properties of an antigen. The term encompasses substanceswhich are immunogenic, i.e., immunogens, as well as substances whichinduce immunological unresponsiveness, or anergy, i.e., anergens.

The antibodies recognize antigens in proteins specific for theinfectious agent and can be detected after the infectious agent has beencleared from the body. Therefore, the detection of an antibody thatrecognizes at least a portion of a protein from an infectious agent isan indication of a current or past infection. In this case, thedetection of antibodies that specifically recognize antigens fromstreptococcal proteins can be used as a marker for a current or paststreptococcal infection. In some embodiments of the above aspects, theanti-streptococcal antibody is selected from the group consisting ofanti-streptolysin 0, anti-deoxyribonuclease-B, anti-hyaluronidase,anti-nicotinamide adenine dinucleotidase and anti-streptokinaseantibodies. In a related embodiment, the anti-streptococcal antibody isan anti-streptolysin 0 antibody.

Methods and assays described herein are directed to the detection ofautoantibodies produced as a result of a streptococcal infection.Autoantibodies following a streptococcal infection can either beanti-streptococcal antibodies that cross-react with neurologicalstructures and recognize an antigen from a protein in a subject ornon-streptococcal antibodies arising as a result of the streptococcalinfection that recognize proteins in the subject. The autoantibodiesdetected by methods and assays described herein recognize an antigenfrom a protein selected from the group consisting of ELAVL2, ELAVL3,ELAVL4, Nova-1, Nova-2, Cdr1, Cdr2 and Cdr3. The term “protein”,“peptide” and “polypeptide” are used interchangeably and in theirbroadest sense to refer to a compound of two or more subunit aminoacids, amino acid analogs or peptidomimetics. The subunits may be linkedby peptide bonds. In another embodiment, the subunit may be linked byother bonds, e.g., ester, ether, etc. A protein or peptide must containat least two amino acids and no limitation is placed on the maximumnumber of amino acids which may comprise a protein's or peptide'ssequence. As used herein the term “amino acid” refers to either naturaland/or unnatural or synthetic amino acids, including glycine and boththe D and L optical isomers, amino acid analogs and peptidomimetics.

In one embodiment of the above aspects of the invention, the protein isselected from the group consisting of ELAVL2, ELAVL3, and ELAVL4. In arelated embodiment, the autoantibody is anti-Hu. The Embryonic LethalAbnormal Vision, Drosophila-Like protein (ELAVL) refers to a family ofRNA-binding proteins also called the Hu family of proteins. Theseproteins are well known, have multiple isoforms, and can be furtherdescribed by Genbank Accession numbers. For example, Genbank AccessionNos. CAI13377.1, CAI13376.1, CAC22160.1, AAH30692.1, EAW58586.1,EAW58585.1, EAW58584.1, EAW58583.1, EAW58582.1, EAW58581.1,NP_(—)001164668.1, NP_(—)001164666.1, NP_(—)004423.2, CAI13378.1, andQ9NZI8.2 are associated with the human ELAVL2 protein. Genbank AccessionNos. AAH11875.1, NP_(—)115657.2, NP_(—)001411.2, Q14576.3, AAK67714.1,AAK57545.1, EAW84221.1, and EAW84220.1 are associated with the humanELAVL3 protein. Genbank Accession Nos. CAI15793.1, CAI15792.1,CAI15791.1, CAI15790.1, CAI15789.1, CAI15788.1, CAI14639.1, CAI14638.1,CAI14637.1, CAI14636.1, AAH36071.1, NP_(—)001138249.1,NP_(—)001138247.1, NP_(—)068771.2, NP_(—)001138246.1, NP_(—)001138248.1,P26378.2, EAX06845.1, EAX06844.1, EAX06843.1, EAX06842.1, EAX06841.1,CAI14635.1, and CAI14634.1 are associated with the human ELAVL4 protein.Each of these Genbank sequences are herein incorporated by reference intheir entirety.

In another embodiment of the above aspects of the invention, the proteinis selected from the group consisting of Nova-1 and Nova-2. In a relatedembodiment, the autoantibody is anti-Ri. The Nova proteins are neuronalRNA binding proteins. These proteins are further described by GenBankAccession Nos. NP_(—)002506.2, NP_(—)006480.2, and NP_(—)006482.1 forthe human Nova-1 protein and 1DTJ_D, 1DTJ_C, 1DTJ_B, 1DTJ_A, Q9UNW9.1,1EC6_B, 1EC6_A, NP_(—)002507.1, AAC72355.1, and AAD13116.1 for the humanNova-2 protein. Each of these Genbank sequences are herein incorporatedby reference in their entirety.

In yet another embodiment of the above aspects of the invention, theprotein is selected from the group consisting of Cdr1, Cdr2 and Cdr3. Ina related embodiment, the autoantibody is anti-Yo. Cdr1, 2, and 3 referto Cerebellar Degeneration Related protein 1, 2, and 3. These proteinsare further described by GenBank Accession Nos. NP_(—)004056.2,P51861.2, AAI13475.1, AAI13473.1, and CAI42370.1 for the human Cdr1protein; NP_(—)001793.1, Q01850.2, AAA51961.1, AAH17503.2, EAW50610.1,and EAW50609.1 for the human Cdr2 protein. Each of these Genbanksequences is herein incorporated by reference in their entirety.

The term “biological sample” as used herein refers to any fluid or solidsample from the body of an animal. Examples of biological samplesinclude but are not limited to plasma, serum, blood, lymphatic fluid,cerebrospinal fluid, synovial fluid, urine, saliva, mucous, phlegm andsputum. In one embodiment, the biological sample is selected from thegroup consisting of blood, serum, and plasma. In another embodiment, thebiological sample is a human sample. A biological sample of the presentinvention may be collected by any suitable method. The biological samplemay be used immediately or may be stored for later use. Any suitablestorage method known in the art may be used to store the biologicalsample; for example the sample may be frozen at about −20° C. to about−70° C.

The term “diagnose” as used herein refers to the act or process ofidentifying or determining a disease or condition in a mammal or thecause of a disease or condition by the evaluation of the signs andsymptoms of the disease or disorder. Usually, a diagnosis of a diseaseor disorder is based on the evaluation of one or more clinical factorsand/or symptoms that are indicative of the disease. That is, a diagnosiscan be made based on the presence, absence or amount of a factor whichis indicative of presence or absence of the disease or condition. Eachfactor or symptom that is considered to be indicative for the diagnosisof a particular disease does not need be exclusively related to theparticular disease; i.e. there may be differential diagnoses that can beinferred from a diagnostic factor or symptom. Likewise, there may beinstances where a factor or symptom that is indicative of a particulardisease is present in an individual that does not have the particulardisease.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 1.0 or 0.1, as appropriate oralternatively by a variation of +/−15%, or alternatively 10% oralternatively 5% or alternatively 2%. It is to be understood, althoughnot always explicitly stated, that all numerical designations arepreceded by the term “about”. It also is to be understood, although notalways explicitly stated, that the reagents described herein are merelyexemplary and that equivalents of such are known in the art.

As used in the specification and claims, the singular form “a”, “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a polypeptide” includes a plurality ofpolypeptides, including mixtures thereof.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but do notexclude others. “Consisting essentially of” when used to definecompositions and methods, shall mean excluding other elements of anyessential significance to the combination for the intended use. Thus, acomposition consisting essentially of the elements as defined hereinwould not exclude trace contaminants from the isolation and purificationmethod and pharmaceutically acceptable carriers, such as phosphatebuffered saline, preservatives, and the like. “Consisting of” shall meanexcluding more than trace elements of other ingredients and substantialmethod steps for administering the compositions of this invention.Embodiments defined by each of these transition terms are within thescope of this invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs.

DETAILED DESCRIPTION OF THE INVENTION

Pediatric Autoimmune Neuropsychiatric Disorder Associated with group AStreptococcal infection (PANDAS) and other post-streptococcal infectioncomplications can occur in subjects, typically children or young adults,after a streptococcal infection. Without being limited to a particulartheory, autoantibodies produced during the immune reaction to theinfection may contribute to the pathogenesis of the disease.Autoantibodies developing following a streptococcal infection, eitheranti-streptococcal antibodies that cross-react with neurologicalstructures or non-streptococcal antibodies arising as a result of thestreptococcal infection that recognize neuronal structures, may causebehavioral changes such as excessive compulsive disorders, tics,abnormal movements, or autism spectrum disorder symptoms.

These disorders are typically difficult to diagnose due to the lack ofconsistent biological markers found in subjects with these diseases.Assays and methods described herein can be used to detect biologicalmarkers present in subjects displaying abnormal behaviors following astreptococcal infection.

Sample Preparation

Provided herein are methods of using the information obtained throughanalysis of the presence or absence of one or autoantibodies in testsamples of acellular biological sample or cell-containing samples. Testsamples may be obtained from an individual or patient. Methods ofobtaining test samples are well-known to those of skill in the art andinclude, but are not limited to, aspirations or drawing of blood orother fluids. Samples may include, but are not limited to, whole blood,serum, plasma, saliva, cerebrospinal fluid (CSF), pericardial fluid,pleural fluid, urine, and eye fluid.

In embodiments in which the presence or absence of an autoantibody willbe determined using an acellular body fluid, the test sample obtainedfrom a person may be a cell-containing liquid or an acellular body fluid(e.g., plasma or serum). In some embodiments in which the test samplecontains cells, the cells may be removed from the liquid portion of thesample by methods known in the art (e.g., centrifugation) to yieldacellular body fluid for the determination of the presence or absence ofcertain autoantibodies.

In other embodiments, the presence or absence of an autoantibody can bedetermined using a cell-containing sample. In these embodiments thecell-containing sample includes, but is not limited to, blood, urine,organ, and tissue samples. Cell lysis may be accomplished by standardprocedures. In certain preferred embodiments, the cell-containing sampleis a whole blood cell lysate. In certain other embodiments, thecell-containing sample is a white blood cell lysate. Methods forobtaining white blood cells from blood are known in the art (Rickwood etal., Anal. Biochem. 123:23-31 (1982); Fotino et al., Ann. Clin. Lab.Sci. 1:131 (1971)). Commercial products useful for cell separationinclude without limitation Ficoll-Paque (Pharmacia Biotech) and NycoPrep(Nycomed).

Identifying the Presence of a Current or Prior Streptococcal Infection

Group A Streptococcus (GAS) is a gram-positive bacterial genus composedof Streptococcus pyogenes strains. Group A Streptococcus strains have asurface antigen recognized by Lancefield serogrouping tests, termed theLancefield Group A antigen. Lancefield groups (there are about 18Lancefield groups) are composed of different Streptococcus speciesgroups that have specific antigens and are distinguished by specificLancefield antibody tests. In addition, group A Streptococcus strainsare beta hemolytic meaning that the bacteria lyse red blood cellssuspended in agar plates with secreted substances. These tests arefrequently used to distinguish group A Streptococcus bacteria from groupB and other Streptococcus groups. These organisms appear as pairs andchains when gram-stained. Although these bacteria can harmlesslycolonize people on their throat and skin, sometimes they can cause mildto serious diseases. Streptococcus bacteria have many components thatcontribute to the pathogens' ability to cause disease:

-   -   Lipoteichoic acid on its surface helps the bacteria to bind to        epithelial cell membranes;    -   M proteins (over 100 types on the GAS bacterial strains) help        the bacteria resist immunologic host defenses;    -   Exotoxins, nucleases and enzymes, for example, DNAses A, C and        D, streptolysin S, proteinase, streptokinase, and pyrogenic        exotoxins (A-D);    -   Human immune system stimulators; for example, streptolysin O,        DNAse B, and hyaluronidase;    -   Capsular polysaccharide (C-substance) composed of a branched        polymer of L-rhamnose and N-acetyl-D-glucosamine may have a role        in increased invasive capacity;    -   C5A peptidase destroys the chemotactic signals by cleaving the        complement component of C5A.

Exotoxins cause the scarlet fever rash, damage organs, may cause shock,and inhibit the human immune system. The M protein, a major virulencefactor, is a macromolecule incorporated in fimbriae present on the cellmembrane projecting on the bacterial cell wall. More than fifty types ofStreptococcus pyogenes M proteins have been identified based onantigenic specificity, and the M protein is the major cause of antigenicshift and antigenic drift among GAS. The M protein binds the hostfibrinogen and blocks the binding of complement to the underlyingpeptidoglycan. This allows survival of the organism by inhibitingphagocytosis. Strains that contain an abundance of M protein resistphagocytosis, multiply rapidly in human tissues, and initiate diseaseprocess. After an acute infection, type-specific antibodies developagainst M protein activity in some cases. R and T proteins are used asepidemiological markers, but have no known role in virulence. Detectionof any protein or antibody produced as a result of or from astreptococcal infection can be used as a marker to determine theexistence of a past or current streptococcal infection.

Methods and assays described herein describe the testing of a biologicalsample for the presence of a marker of a streptococcal infection. Themarker can indicate a live bacterial infection or the presence of a pastinfection. The term “live bacterial infection” is intended to mean abacterial infection that is still present in the subject. Paststreptococcal infections can be diagnosed by determining the presence orabsence of a marker of a streptococcal infection that remains after thebacteria have been cleared from the body. A live streptococcal infectionor the existence of a past streptococcal infection can be diagnosed by avariety of methods known in the art. For example, after a history andphysical examination, many clinicians presumptively diagnose strepthroat from its symptom production and throat appearance. Astreptococcal infection can also be diagnosed from cultures of thethroat or other site of infection. Cultures can be tested on blood agarplates that contain two different antibiotics and cause beta hemolysis(complete blood red cell lysis to form a clear area) of the red bloodcells. In addition, there are rapid tests (RADT or rapid antigendetection test) that take only a few minutes to complete that detect agroup A carbohydrate surface antigen produced by the streptococcalbacteria, with specificity of about 95% or better and fairly goodsensitivity of about 80%-90%.

A streptococcal infection can also be diagnosed by testing for thepresence or absence of streptococcal antibodies in a biological sample.Suitable biophysical or biomolecular detection methods for qualitativelydetecting an antibody comprise any suitable method known in the art.Such methods include, without being limited thereto, methods as appliedfor qualitative or quantitative assays such as, for example,Enzyme-linked Immunosorbent Assay (ELISA), ELISPOT-Assay, Western-Blotor Immunoassays. In one embodiment, the presence or absence of anantibody selected from the group consisting of anti-streptolysin O,anti-deoxyribonuclease-B, anti-hyaluronidase, anti-nicotinamide adeninedinucleotidase and anti-streptokinase is determined. In some instances,it may be more desirable to test for the presence of more than oneantibody to confirm the presence or past existence of a streptococcalinfection in the subject. In one embodiment, the presence or absence ofanti-streptolysin O is used as a marker to test for a current or paststreptococcal infection. Detection of antibodies as described herein canalso be determined by methods the same as those used for thecharacterization of autoantibodies in a sample in foregoingdescriptions.

Characterization of Autoantibodies in a Sample

Autoantibodies and/or antibodies can be detected in a sample by avariety of known methods. As described previously, these methodsinclude, for example, Enzyme-linked Immunosorbent Assay (ELISA),ELISPOT-Assay, Western-Blot or Immunoassays. Such methods may compriseoptical, radioactive, chromatographic methods, fluorescence detectionmethods, radioactivity detection methods, Coomassie-Blue staining,Silver staining or other protein staining methods, electron microscopymethods, methods for staining tissue sections by immunohistochemistry orby direct or indirect immunofluorescence, etc. Such methods may beapplied either with the autoantibody or may involve the use of furthertools, for example, the use of a secondary antibody, specificallybinding to the constant part of the autoantibody. The secondary antibodymay be labeled to allow a specific detection of the secondary antibody.

Immunoassays, such as an ELISA are commonly used for the detection ofantibodies in a biological sample. In one example of an ELISA, theantibodies or antigens are immobilized on a selected surface, such as awell in a polystyrene microtiter plate, dipstick, or column support.Then, a test composition suspected of containing the desired antigen orantibody, such as a biological sample, is added to the wells. Afterbinding and washing to remove non specifically bound immune complexes,the bound antigen or antibody may be detected. Detection is generallyachieved by the addition of another antibody, specific for the desiredantigen or antibody that is linked to a detectable label. This type ofELISA is known as a “sandwich ELISA.” Detection also may be achieved bythe addition of a second antibody specific for the desired antigen,followed by the addition of a third antibody that has binding affinityfor the second antibody, with the third antibody being linked to adetectable label. Variations on ELISA techniques are known to those ofskill in the art.

As used herein, the term “label” intends a directly or indirectlydetectable compound or composition that is conjugated directly orindirectly to the composition to be detected, for example, N-terminalhistadine tags (N-His), magnetically active isotopes, e.g., ¹¹⁵Sn, ¹¹⁷Snand ¹¹⁹Sn, a non-radioactive isotopes such as ¹³C and ¹⁵N,polynucleotide or protein such as an antibody so as to generate a“labeled” composition. The term also includes sequences conjugated tothe polynucleotide that will provide a signal upon expression of theinserted sequences, such as green fluorescent protein (GFP) and thelike. The label may be detectable by itself (e.g. radioisotope labels orfluorescent labels) or, in the case of an enzymatic label, may catalyzechemical alteration of a substrate compound or composition which isdetectable. The labels can be suitable for small scale detection or moresuitable for high-throughput screening. As such, suitable labelsinclude, but are not limited to magnetically active isotopes,non-radioactive isotopes, radioisotopes, fluorochromes, chemiluminescentcompounds, dyes, and proteins, including enzymes. The label may besimply detected or it may be quantified. A response that is simplydetected generally comprises a response whose existence merely isconfirmed, whereas a response that is quantified generally comprises aresponse having a quantifiable (e.g., numerically reportable) value suchas an intensity, polarization, and/or other property. In luminescence orfluorescence assays, the detectable response may be generated directlyusing a luminophore or fluorophore associated with an assay componentactually involved in binding, or indirectly using a luminophore orfluorophore associated with another (e.g., reporter or indicator)component. Examples of luminescent labels that produce signals include,but are not limited to bioluminescence and chemiluminescence. Detectableluminescence response generally comprises a change in, or an occurrenceof, a luminescence signal. Suitable methods and luminophores forluminescently labeling assay components are known in the art anddescribed in, for example, Haugland, Richard P. (1996) Handbook ofFluorescent Probes and Research Chemicals (6^(th) ed.). Examples ofluminescent probes include, but are not limited to, aequorin andluciferases.

Competition ELISAs are assays in which test samples compete for bindingwith known amounts of labeled antigens or antibodies. The amount ofreactive species in the unknown sample is determined by mixing thesample with the known labeled species before or during incubation withcoated wells. The presence of reactive species in the sample acts toreduce the amount of labeled species available for binding to the welland thus reduces the ultimate signal. Irrespective of the formatemployed, ELISAs have certain features in common, such as coating,incubating or binding, washing to remove non specifically bound species,and detecting the bound immune complexes.

Antigen or antibodies may also be linked to a solid support, such as inthe form of plate, beads, dipstick, membrane, or column matrix, and thesample to be analyzed is applied to the immobilized antigen or antibody.In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period. The wells of theplate will then be washed to remove incompletely-adsorbed material. Anyremaining available surfaces of the wells are then “coated” with anonspecific protein that is antigenically neutral with regard to thetest antisera. These include bovine serum albumin (BSA), casein, andsolutions of milk powder. The coating allows for blocking of nonspecificadsorption sites on the immobilizing surface and thus reduces thebackground caused by nonspecific binding of antisera onto the surface.

In the method of the present invention for detecting the presence of atleast one antibody against one of the above defined autoantigens orantigens in a sample a qualitative or a quantitative determination canbe carried out. “Qualitative determination” in the context of theinventive method is to be understood as any method for specificallyidentifying the presence of a specific autoantibody, for example, anautoantibody directed against one or more of specific proteins selectedfrom the autoantigenic proteins ELAVL2, ELAVL3, ELAVL4, Nova-1, Nova-2,Cdr1, Cdr2 and Cdr3, or a fragment, variant or epitope thereof.“Quantitative determination” in the context of the inventive method isto be understood as any method for determination of an antibody or(antibody) proteins or peptides, protein fragments, variants or epitopesthereof, known by a skilled person suitable for quantifying the amountof a autoantibody or a secondary antibody, in a sample. As an example,the inventive method may be carried out with a test sample as aconcurrent standard, containing a defined amount of an autoantibodyagainst at least one of the above autoantigenic proteins, and inparallel with a second sample, which is derived from a patient andcontains an unknown amount of an autoantibody to be determined againstat least one of the above autoantigenic proteins. A comparison of thedefined amount of the autoantibody in the test sample with the amount ofthe autoantibody in the second sample will allow a precise determinationof the amount of autoantibody in the second sample. A concurrentstandard may be applied either parallel to carrying out the inventivemethod or, for example, prior to said method, by preparing a standardcurve, which may be used in the subsequent quantification.

Testing the Reactivity of Autoantibodies to Neuronal Cells

Autoantibodies produced in an immune response to a streptococcalinfection can be specific to proteins described herein or cancross-react to the proteins described herein, for example, a proteinselected from the group consisting of ELAVL2, ELAVL3, ELAVL4, Nova-1,Nova-2, Cdr1, Cdr2 and Cdr3. These proteins can be expressedendogenously in neuronal cells and can cross-react with autoantibodiesproduced from a streptococcal infection. The term “react” in this senseis intended to describe the binding of the autoantibody to a neuronalcell, structure, nucleic acid, nucleoprotein, or protein. Suchreactivity can be detected by a variety of common methods.

In one embodiment, the reactivity of an autoantibody to a neuronal cellcan be determined by immunoassays. In a related embodiment, theimmunoassay is an immunofluorescence assay. In a further embodiment, theimmunofluorescence assay is an indirect immunofluorescence assay.Immunofluorescence is a technique used for light microscopy with afluorescence microscope and is used primarily on biological samples.This technique uses the reactivity of antibodies to their antigen totarget fluorescent dyes to specific biomolecule targets within a cell,and therefore allows visualization of the distribution of the targetmolecule through the sample. Immunofluorescence is a widely used exampleof immunostaining and is a specific example of immunohistochemistry thatmakes use of fluorophores to visualize the location of the antibodies.

Immunofluorescence can be used on tissue sections, cultured cell lines,or individual cells, and may be used to analyze the distribution ofproteins, glycans, small biological and non-biological molecules, andthe reactivity of antibodies in a biological sample to cells.Immunofluorescence can be used in combination with other, non-antibodymethods of fluorescent staining, for example, use of DAPI to label DNAor Evans blue stain to label cells. Several microscope designs can beused for analysis of immunofluorescence samples; the simplest is theepifluorescence microscope, and the confocal microscope is also widelyused. Various super-resolution microscope designs that are capable ofmuch higher resolution can also be used.

Primary, or direct, immunofluorescence uses a single antibody that ischemically linked to a fluorophore. The antibody recognizes the targetmolecule and binds to it, and the fluorophore it carries can be detectedvia microscopy.

Secondary, or indirect, immunofluorescence uses two antibodies; theunlabeled first (primary) antibody specifically binds the targetmolecule, and the secondary antibody, which carries the fluorophore,recognizes the primary antibody and binds to it. Multiple secondaryantibodies can bind a single primary antibody. This provides signalamplification by increasing the number of fluorophore molecules perantigen.

This protocol is possible because an antibody consists of two parts, avariable region (which recognizes the antigen) and constant region(which makes up the structure of the antibody molecule). It is importantto realize that this division is artificial and in reality the antibodymolecule is four polypeptide chains: two heavy chains and two lightchains. Various primary antibodies can recognize various antigens (havedifferent variable regions), but all share the same constant region. Allthese antibodies may therefore be recognized by a single secondaryantibody.

Different primary antibodies with different constant regions aretypically generated by raising the antibody in different species. Forexample, a researcher might create primary antibodies in a goat thatrecognize several antigens, and then employ dye-coupled rabbit secondaryantibodies that recognize the goat antibody constant region (“rabbitanti-goat” antibodies). The researcher may then create a second set ofprimary antibodies in a mouse that could be recognized by a separate“goat anti-mouse” secondary antibody. This allows re-use of thedifficult-to-make dye-coupled antibodies in multiple experiments.

In some instances, the reactivity of an autoantibody with a neuronalcell can be detected by a physical change in the neuronal cells. In oneembodiment, this physical change is neurite retraction. In a neuriteretraction assay, the change in the length of neurites of neuronal cellscan be determined after addition of a biological sample withautoantibodies. A decrease in the length of neurites after theadministration of an autoantibody is an indication of reactivity of theautoantibody to the neuronal cell. This assay is typically performed onneuronal cells growing in a cell culture environment. Techniques forculturing and differentiating neuronal cells are known in the art.

Diagnosis of Disease

Provided herein are methods and assays for the diagnosis of certaindisease that can occur following a group A streptococcal infection. Byway of example, these diseases include PANDAS, post-GABHSglomerulonephritis, rheumatic fever, Syndenham's chorea, autism spectrumdisorder, obsessive-compulsive disorder, Tourette syndrome, and ticdisorders. These disorders usually occur in children and can also beassociated with exacerbations such as emotional lability, enuresis,anxiety, and deterioration in handwriting. In one embodiment of theaspects described herein, the disease is PANDAS.

The aforementioned assays and methods can be used to diagnose disease ina subject. A subject can be a mammal such as a human or laboratoryresearch mammal such as a mouse, a rat, or a rabbit. In one embodiment,the subject is a human. The subject can also be a human patient underthe care of a medical practitioner.

Example 1 Hu, Ri, and Yo Antibody Immunofluoresence Assay

Biological samples can first be obtained and tested foranti-streptococcal antibodies such as anti-streptolysin O. Eightyanti-streptolysin O (ASO) positive serum samples, 30 ASO negative serumsamples, and ten normal serum samples were obtained. For thedetermination of ASO positive or negative, an antibody quality of 200IU/ml was used. Samples were submitted for ASO testing without clinicalinformation and used after de-identification.

1:40 dilutions of the patient sera was prepared in a pH bufferedsolution. Slides with monkey cerebellar tissues affixed thereto wereremoved from cold storage and kept at room temperature for five minutes.Approximately 50 μl of diluted sera and controls were applied to thereaction wells on the slides. The controls consisted of samples withantibodies determined to have reactivity to one or all of the Hu, Yo,and Ri antigens. The slides were incubated for 30 minutes at roomtemperature in a humidity chamber. Next, the slides were rinsed in apH-buffered solution and placed in a washing dish with the pH-bufferedsolution for two minutes. The slides were then removed from the washingdish and the edges of the slides were blotted with a paper towel.Approximately one drop of a diluted secondary conjugate was then addedto each well. The slides were incubated at room temperature for 30minutes in a humidity chamber. Next the slides were rinsed with apH-buffered solution and placed in a washing dish filled with thepH-buffered solution and one drop of Evans blue stain. The slides wereincubated in this solution for 2-5 minutes and then washed with freshpH-buffered solution.

Twelve samples showed fluorescence in IFA up to at least 1:160 titer.These samples were then tested in Euroimmune Anti-Neuronal AntigensWesternblot (IgG) assay, along with four samples that were negative inthe immunofluoresence assay. These results are depicted in the followingtable.

ASO Result Western Blot (200 IU/ml ASO Value IFA Result Ri, Yo, Ri, Hu,Yo, Sample cutoff) (IU/ml) (1:40 screen) 80 kDa 62 kDa 55 kDa 38 kDa 34kDa A NT (Normal) n/a + (1:320) B − 49 + (1:160) (+) C + 265 + (1:320)D + 331 + (1:160) E + 778 + (1:320) + + (+) F − 65 + (1:160) + G + 213 +(1:160) (+) H − 21 + (1:160) I + 501 + (1:320) J + 319 + (1:160) + K +262 + (1:320) + + (+) L − 6 + (1:160) M + 263 — (+) N + 581 — O + 441 —(+) P + 498 — +

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1. A method for diagnosing a disease in a subject, said methodcomprising, (a) determining the presence or absence of one or moreautoantibodies in a biological sample from the subject with a previousStreptococcal infection, wherein the one or more autoantibodiesrecognize an antigen from a protein selected from the group consistingof ELAVL2, ELAVL3, ELAVL4, Nova-1, Nova-2, Cdr1, Cdr2, and Cdr3 and (b)identifying the subject as (i) having a disease selected from the groupconsisting of PANDAS, post-GABHS glomerulonephritis, rheumatic fever,autism and Syndenham's chorea, when the presence of one or moreantibodies is detected, or (ii) not having one of said diseases causedby the Streptococcal infection when each of said antibodies is absent.2. (canceled)
 3. The method of claim 1 wherein at least one autoantibodyis selected from the group consisting of anti-Hu, anti-Ri and anti-Yo.4-7. (canceled)
 8. The method of claim 1 wherein the disease is PANDAS.9-10. (canceled)
 11. A method comprising: (a) testing a biologicalsample from a subject for the presence of a marker of a streptococcalinfection; and (b) testing the biological sample for the presence orabsence of one or more autoantibodies that recognize an antigen from aprotein selected from the group consisting of ELAVL2, ELAVL3, ELAVL4,Nova-1, Nova-2, Cdr1, Cdr2 and Cdr3.
 12. The method of claim 11 whereinstep (a) comprises determining the presence or absence of one or moreanti-streptococcal antibodies.
 13. The method of claim 12 wherein theone or more anti-streptococcal antibodies are selected from the groupconsisting of anti-streptolysin O, anti-deoxyribonuclease-B,anti-hyaluronidase, anti-nicotinamide adenine dinucleotidase andanti-streptokinase antibodies. 14-15. (canceled)
 16. The method of claim11 wherein at least one autoantibody is selected from the groupconsisting of anti-Hu, anti-Ri and anti-Yo. 17-22. (canceled)
 23. Amethod comprising: (a) testing a biological sample from a subject forthe presence of a marker of a streptococcal infection; and (b) testingthe reactivity of one or more autoantibodies in the sample to neuronalcells wherein the one or more autoantibodies recognize an antigen from aprotein selected from the group consisting of ELAVL2, ELAVL3, ELAVL4,Nova-1, Nova-2, Cdr1, Cdr2 and Cdr3.
 24. The method of claim 23 whereinthe reactivity is determined by an indirect immunofluoresence assay. 25.The method of claim 23 wherein the reactivity is determined by neuriteretraction.
 26. The method of claim 23 wherein step (a) comprisesdetermining the presence or absence of one or more anti-streptococcalantibodies.
 27. The method of claim 26 wherein the one or moreanti-streptococcal antibodies are selected from the group consisting ofanti-streptolysin O, anti-deoxyribonuclease-B, anti-hyaluronidase,anti-nicotinamide adenine dinucleotidase and anti-streptokinase. 28.(canceled)
 29. The method of claim 23 wherein the protein is selectedfrom the group consisting of ELAVL2, ELAVL3, and ELAVL4.
 30. The methodof claim 29 wherein at least one autoantibody is anti-Hu.
 31. The methodof claim 23 wherein the protein is selected from the group consisting ofNova-1 and Nova-2.
 32. The method of claim 31 wherein at least oneautoantibody is anti-Ri.
 33. The method of claim 23 wherein the proteinis selected from the group consisting of Cdr1, Cdr2, and Cdr3.
 34. Themethod of claim 33 wherein at least one autoantibody is anti-Yo. 35-36.(canceled)